Isolation and biomimetic synthesis of (±)-calliviminones A and B, two novel Diels-Alder adducts, from Callistemon viminalis

Article abstract of DOI:10.1016/j.tetlet.2014.11.075

(±)-Calliviminones A (1) and B (2), two Diels-Alder adducts of polymethylated phloroglucinol and myrcene with unprecedented spiro-[5.5] undecene skeleton, were isolated from the fruits of Callistemon viminalis. Structural elucidation was accomplished by NMR spectra studies, and the biomimetic synthesis of compounds 1 and 2 confirmed the pivotal role of Diels-Alder reaction in the plausible biosynthetic pathway. Compounds 1 and 2 were also the first example of Carbon Diels-Alder adducts between phloroglucinol and terpene. Bioactivity scan indicated that 1 and 2 showed moderate inhibition on NO production on lipopolysaccharide-induced RAW264.7 macrophages.

Full text of DOI:10.1016/j.tetlet.2014.11.075

Tetrahedron Letters 56 (2015± 229–232
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Isolation and biomimetic synthesis of (± ±)calliꢀiminones A and B, two
noꢀel Diels–Alder adducts, from Callistemon viminalis
Lin Wu a, , Jun Luo
a,b,
, Yalong Zhang , Mengdi Zhu , Xiaobing Wang , Jianguang Luo a,b,
a
a
a,b
a,b
a
a
a
a
a
a
Minghua Yang , Boyang Yu , Hequan Yao , Yue Dai , Qinglong Guo , Yijun Chen , Hongbin Sun ,
a,b,
⇑
Lingyi Kong
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
(± ±-Calliviminones A (1± and B (2±, two Diels–Alder adducts of polymethylated phloroglucinol and myr-
cene with unprecedented spiro-[5.5] undecene skeleton, were isolated from the fruits of Callistemon vim-
inalis. Structural elucidation was accomplished by NMR spectra studies, and the biomimetic synthesis of
compounds 1 and 2 confirmed the pivotal role of Diels–Alder reaction in the plausible biosynthetic path-
way. Compounds 1 and 2 were also the first example of Carbon Diels–Alder adducts between phloroglu-
cinol and terpene. Bioactivity scan indicated that 1 and 2 showed moderate inhibition on NO production
on lipopolysaccharide-induced RAW264.7 macrophages.
Received 10 October 2014
Revised 13 November 2014
Accepted 16 November 2014
Available online 21 November 2014
Keywords:
b-Triketones
Diels–Alder reaction
Biomimetic synthesis
Spiro compounds
Ó 2014 Elsevier Ltd. All rights reserved.
Naturally occurring adducts of acylphloroglucinol and terpene
constitute a large family of natural products that display diverse
molecular architectures and a wide range of biological profiles,
such as antibacterial, insecticidal and antiproliferative proper-
based on the hypothesis of biosynthetic pathway, which confirmed
the pivotal role of Diels–Alder reaction in the biosynthetic pathway.
Bioactivity screen showed that compounds 1 and 2 had inhibition
on nitric oxide (NO± production on lipopolysaccharide-induced
1
–3
ties.
Callistemon viminalis (Myrtaceae± is a shrub native to Aus-
RAW264.7 macrophages with IC50 of 29.9 and 28.8 lM, respec-
tralia, which has also been cultivated in the south of China and
used for treatment of cold and arthralgia in Chinese folk medicine.
tively. Herein, we reported the isolation, structural elucidation, bio-
mimetic synthesis, and inhibitory activities on NO production.
Compound 1 was isolated as colorless gum, and its molecular
4
Previous researches indicated that polymethylated phloroglucinols
5
,6
by its [M+H]+ quasi-
, 373.2737± in
are prominent secondary metabolites of the genus Callistemon.
formula was determined to be C24
36 3
H O
As a part of our research program to discover natural products
with a unique molecular architecture in Myrtaceae family, two
novel adducts of polymethylated phloroglucinol and acyclic mono-
terpene (myrcene± possessed an unprecedented spiro-[5.5] unde-
cene skeleton, named (± ±-calliviminones A and B, were isolated
from the fruits of C. viminalis collected in Guangdong province of
China. The unique spiro skeleton was likely formed via a [4+2]-
molecular ion at m/z 373.2739 (calcd for C24H O
37 3
ꢀ1
the HRESIMS. The absorption bands at 1699 and 1645 cm in IR
spectrum indicated the presence of carbonyl and doubled bond
1
13
groups. Consideration of the H and C NMR spectra in conjunc-
tion with information from HSQC spectrum, the presence of an iso-
butyl syncarpic acid unit could be approved by the following
characteristic resonance: d
0.83; d 213.0, 208.8, 208.7, 67.1, 56.9, 56.6, 41.4, 30.2, 26.4,
26.1, 25.2, 24.9, 24.2, 19.2. This conclusion was also confirmed
by the key HMBC correlations (Fig. 2± of H-8 (d 1.68± with C-9
(d 24.2± and C-7 (d 41.4±; H-7 (d 2.18± with C-6 (d 67.1±; Me-
13 (d 1.39± with C-1 (d 208.7± and C-6 (d 67.1± and Me-12 (d
.38± with C-5 (d 208.8± and C-6 (d 67.1±. The remaining 10 car-
H
1.41, 1.39, 1.38, 1.34, 1.68, 2.18, 0.88,
6
(7±
cycloaddition between
D
of isobutyl syncarpic acid moiety and
C
0
0
0
0
1
(2 ± 3 (4 ±
6(7±
6,7
D
,
of myrcene unit in 1, and those were between
D
0
0 0 0
(3 ± 2 (1 ±
4
and
D
,
in 2, which were the first example of Diels–Alder
H
adducts between phloroglucinol and terpene units in Carbon
Diels–Alder manner. Both 1 and 2 were synthesized successfully
C
C
H
C
H
C
C
H
1
C
C
bon atoms were classified to be two methyl groups, four methyl-
enes, and four olefinic carbons based on the HSQC correlations,
which may formed a monoterpene moiety. Considering the 24
⇑
Corresponding author. Tel./fax: +86 25 83271405.
E-mail address: cpu_lykong@126.com (L. Kong±.
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.tetlet.2014.11.075
040-4039/Ó 2014 Elsevier Ltd. All rights reserved.
0

2
30
L. Wu et al. / Tetrahedron Letters 56 (2015) 229–232
carbon atoms and 7° of unsaturation in the structure of 1, together
with the number of methyls, it could be speculated that compound
tion. That was also confirmed by the obviously downfield shift of
0
H-1
a
affected by the negative shielding effect of the olefinic bond
1
was an adduct of polymethylated phloroglucinol with
and two carbonyls at C-1 and C-5. Aforementioned information
monoterpene.
indicated that the cyclohexene ring adopted
conformation.
Compound 2, colorless gum, has the same molecular formula as
that of 1 established by HRESIMS. Similar NMR data (Table 1±,
a half chair
0
0
The presence of an isopentene moiety (C-6 to C-10 ± was deter-
0
0
mined by the cross peaks between H-7 (d
H
5.05± and C-9 (d
2.07± and C-5 (d
C
25.8±
0
0
0
0
and C-10 (d
C
17.8±; H-6 (d
H
C
37.2± and C-7 (d
C
0
0
1
24.3± in the HMBC spectrum. The fragments of C-3 to C-6 and
0
0
1
1
C-1 to C-4 were established by the H– H COSY and the HMBC
spectra (Fig. 2±, which indicated that the monoterpene moiety of
Table 1
1
H (500 MHz± and 13C (125 MHz± NMR data of 1 and 2 in CDCl
3
1
possessed myrcene skeleton. The key HMBC correlations
0
between these two parts, H-1
a
(d
H
2.48± to C-6 (d
28.0± and C-3 (d
C
67.1± and C-1
138.4±, indi-
No.
1
2
0
0
(d
C
208.7±, H-7 (d
H
2.18± to C-4 (d
C
C
d
H
(mult; J, Hz±
d
C
d
H
(mult; J, Hz±
d
C
cated that the isobutyl syncarpic acid and the myrcene moieties
1
2
3
4
5
6
7
8
9
1
1
1
1
208.7
56.6
213
208.9
56.4
213.2
56.3
208.9
68.8
40.5
29.7
24.3
19
26.3
24.4
26.1
26.6
24.1
0
0
were connected through C-1 to C-6 and C-4 to C-7 bonds, which
0
was also confirmed by the correlations of H-7 with H-4 and of
0
0
1
1
H-1 with H-2 in the H– H COSY spectrum (Fig. 2±. Thus, the pla-
nar structure of 1 was demonstrated as shown in Figure 1, which
possessed spiro-[5.5] undecene skeleton constructed of an isobutyl
syncarpic acid and a myrcene moieties.
56.9
208.8
67.1
41.4
30.2
24.2
19.2
26.4
24.9
25.2
26.1
31.5
a
a
2.07, ma
1.58, m
0.88, d (7.0±
0.79, d (7.0±
1.44, s
1.38, s
1.40, s
1.33, s
2.18, m
1.68, m
a
Due to the optical rotation measured to be zero, 1 was a racemic
mixture of enantiomers with the stereogenic center at C-7 and the
relative configuration of compound 1 was established through
ROESY spectrum (Fig. 2±. Cross peaks from Me-10 to Me-12 and
Me-9 to Me-13 indicated that the above protons were on the same
side and H-7 was located at opposite side and adopted as b-config-
0.88, d (7.0±
0.83, d (7.0±
1.41, s
0
1
2
3
1.38, s
1.39, s
1.34, s
14
0
1
a
2.48, dd (17.5, 3.0±
2.21, d (17.5±
0
a
a
0
0
1 b
2.14, m
1.98, m
uration. The ROESY correlation from H-1
a
H
(d
H H
2.48± to H-4 a (d
0
2
5.26, br s
115.4
138.4
28
5.35, br s
119.3
133.7
31.3
0
2
.23± and Me-14 indicated that H-1
a
(d
2.48± was present on
0
3
the underside of the cyclohexene ring and adopted as
a-configura-
0
0
0
0
0
0
0
2.23,ma
4
4
5
6
7
8
9
1
a
b
2.49, dd (17.5, 1.5±
a
a
2.00, m
2.06, m
a
a
1.97, m
37.2
26.4
124.3
131.6
25.8
17.8
1.98, m
37.6
26.2
124.3
131.7
25.8
17.8
a
a
2.07, m
2.10, m
5.05, t (7.0±
5.07, tt (7.0, 1.5±
1.67, s
1.59, s
1.66, s
1.59, s
0
0
a
Singal pattern unclear due to overlapping.
Figure 1. Structures of compounds 1 and 2.
Figure 2. Key HMBC (?±, 1H– H COSY (—± and ROESY (M± correlations of 1.
1
Scheme 1. The plausible biogenetic pathway for 1 and 2.

L. Wu et al. / Tetrahedron Letters 56 (2015) 229–232
231
Scheme 2. Biomimetic Synthesis of compounds 1 and 2.
properties, and the same molecular formula, suggested that 2 was
available phloroglucinol (3± and myrcene (9± as substrate
(Scheme 2±. The key intermediate isobutylidene syncarpic acid
(8± was arisen from phloroglucinol (3± through acetylation, meth-
0
an isomer of 1. The obvious difference of the chemical shift for C-1 ,
0
0
0
C-2 , and C-3 between 1 and 2 was observed. The signal for C-1
0
was upfield shifted from d
115.4 downfield shifted to d
upfield shifted to d
C
31.5 in 1 to d
C
24.1 in 2, for C-2 from
ylation, an acid induced retro-Claisen condensation and Knoevena-
0
13–15
d
C
C
119.3 and for C-3 from d
C
138.4
gel condensation.
Since 8 was prone to isomerization, it was
16
C
133.7. Above similarities and differences indi-
immediately reacted with the myrcene (9± in toluene at 110 °C.
cated that the isobutyl syncarpic acid conjugated with myrcene in
On the basis of electronic effect and steric effect, the C-7 in isobu-
0
different modes in 2. In the HMBC spectrum, H-4
H
a (d 2.49± had
tylidene syncarpic acid as electron-withdrawing, is prone to react
0
correlations with C-1 (d
C
208.9± and C-6 (d
C
68.8± in 2 instead of
2.21± had cross peaks with C-8 (d
H
2.23± in 1, which indicated that the
with the C-4 in myrcene as electron-donating. After separation
0
0
H-1
a
(d
H
2.48± in 1, H-1 (d
H
C
and purification, two target products were obtained with a ratio
of 6:1 and 35% yield successfully, whose structures were deter-
mined to be 1 and 2 by identical consistent t and UV in HPLC,
R
0
2
9.7± in 2 instead of H-4 (d
cyclohexene ring in 2 formed between d
more, this deduction was also confirmed by the key HMBC correla-
0 0 0 0
4 (3 ± 2 (1 ±
6(7±
and d
,
. Further-
HRESIMS, and 1D NMR. Compounds 10 and 11, two main by-prod-
ucts originated from the isomerization of 8, were also obtained in
about 45% yield, which lead to the low chemical yield of com-
pounds 1 and 2. In conclusion, our study provides strong evidence
for the proposed biosynthetic pathways of these two natural
products.
Compounds 1 and 2 were screened for inhibition of NO produc-
tion. MTT assay revealed no obvious cytotoxic effects (over 90% cell
survival± of cells treated with the compound at concentrations up
0
0
0
tions of H-5 (d
C-8 (d
and H-1 (d
H
1.98± with C-4 (d
29.7±, and H- H COSY correlation between H-2 (d
C
31.3±, H-1 (d
H
2.21, 1.98± with
1
1
0
C
H
5.35±
0
H
2.21, 1.98±. Thus, the planar structure of 2 was dem-
onstrated as shown in Figure 1, which also possessed a spiro-
5.5] undecene skeleton formed between C-6 and C-7 of isobutyl
[
0
0
syncarpic acid and C-4 and C-1 of myrcene moiety. The same
ROESY correlations with 1 and zero of optical rotation indicated
that 2 was also a racemic mixture of enantiomers and had the same
relative configuration as that of 1. Thus, the structure of 2 was
established as shown in Figure 1.
to 100
vated RAW 264.0 cells, with an IC50 value of 29.9 and 28.8
respectively, comparable to that of the positive control N-mono-
lM. Compounds 1 and 2 showed NO inhibition on LPS-acti-
lM,
The plausible biosynthetic pathway for 1 and 2 was depicted in
Scheme 1. According to the hypothesis, 1 and 2 were derived from
isobutylidene syncarpic acid and myrcene via a [4+2]-cycloaddi-
tion under the catalysis of enzyme or UV.8 In turn, isobutylidene
1
7
methyl-L-arginine at 40.45 lM. The result suggests that this kind
of skeleton may be a potential candidate for anti-inflammatory
agent.
9
,10
syncarpic acid could be generated from flavesone (b-triketones±
via reduction and dehydration. Notably, isobutylidene syncarpic
Acknowledgments
11
acid was prone to transformed into G
3
,
which was termed as
G-regulators exercise control over a wide range of physiological
functions in plants. In view of the fact that both 1 and 2 were iso-
lated as racemic mixture with the stereogenic center at C-7, the C–
C bond formation should be an enzyme-catalyzed without stere-
oselectivity process.
This research work was financially supported by the National
Natural Sciences Foundation of China (21272275, 81202899±, the
Youth Fund Project of Basic Research Program of Jiangsu Province
(Natural Science Foundation, BK2012351±, the Program for New
Century Excellent Talents in University (NCET-2013-1035±, the
Program for Changjiang Scholars and Innovative Research Team
in University (PCSIRT-IRT1193±, and the Project Funded by the Pri-
ority Academic Program Development of Jiangsu Higher Education
Institutions (PAPD±.
However, the different structures and ratios of 1 and 2 could be
attributed to the regioselectivity in [4+2]-cycloaddition. Isobuty-
lidene syncarpic acid possessed
a,b-unsaturated carbonyl moiety,
with open-chain carbon-dienes, are generally highly ortho–para
regioselective because the oxygen complexation increases the dif-
1
2
ference of LUMO coefficients of the alkene moiety. Myrcene bear-
Supplementary data
0
0
ing a EDG at C3 has its largest HOMO coefficient at C4 , while
isobutylidene syncarpic acid has the largest LUMO coefficient at
C7. Paring these two coefficients gives 1 as a major regioisomer.
To validate above biosynthetic hypothesis, a biomimetic syn-
thesis of compounds 1 and 2 was conducted with commercially
Supplementary data (detailed description of the experimental
procedures, copies of the HR-ESI-MS, 1D and 2D NMR spectra of
1
and 2± associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.tetlet.2014.11.075. These data

2
32
L. Wu et al. / Tetrahedron Letters 56 (2015) 229–232
include MOL files and InChiKeys of the most important compounds
described in this article.
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